Assays for mammalian tyrosinase: a comparative study
This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed.
- Research Organization:
- Universidad de Murcia (Spain)
- OSTI ID:
- 5905058
- Journal Information:
- Pigment Cell Res.; (United States), Vol. 1:5
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
MELANIN
BIOSYNTHESIS
TYROSINASE
ENZYME ACTIVITY
COMPARATIVE EVALUATIONS
EXPERIMENTAL NEOPLASMS
FRACTIONATION
ISOENZYMES
ISOTOPE DILUTION
MELANOMAS
MICE
MICROSOMES
OXIDOREDUCTASES
RADIOASSAY
SPECTROPHOTOMETRY
TYROSINE
AMINO ACIDS
ANIMALS
CARBOXYLIC ACIDS
CELL CONSTITUENTS
DISEASES
ENZYMES
HYDROXY ACIDS
HYDROXY COMPOUNDS
HYDROXYLASES
ISOTOPE APPLICATIONS
MAMMALS
NEOPLASMS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
PIGMENTS
RODENTS
SEPARATION PROCESSES
SYNTHESIS
TRACER TECHNIQUES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques